ESGP and CLC/Gal-10 levels present in EST samples and biopsy extracts were determined using commercial (EDN, ECP; MBL International, Woburn, MA, USA) or inhouse developed (MBP1, CLC/Gal-10, EPX) ELISAs. Assays for EDN and ECP were performed according to the manufacturer's instructions. Double-antibody sandwich ELISAs for MBP1 and CLC/Gal-10 were developed and standardised at the Ackerman laboratory (Chicago, IL, USA) and for EPX at the Lee laboratory (Scottsdale, AZ, USA). MBP1 and CLC/Gal-10 ELISAs used standard curves generated using the purified eosinophil-derived protein, prepared as previously described.7 (link)
20–22 For MBP1, mouse monoclonal antibodies were used for both capture and detection (mouse anti-human MBP1-clones, J14-8A2 and J13-6B6, respectively, kindly provided by Dr Hirohito Kita, Mayo Clinic, Rochester, Minnesota, USA), the detection antibody being biotinylated and detected using streptavidin-conjugated horseradish peroxidase (ExtraAvidin Peroxidase, E2886; Sigma-Aldrich, St. Louis, Missouri, USA) and FAST OPD Tablet substrate (P9187; Sigma-Aldrich). CLC/Gal-10 ELISAs used a mouse monoclonal capture antibody (Anti-human Gal-10/CLC, Cell Sciences, Canton, Massachusetts, USA), whereas the detection antibody was a CLC/Gal-10 affinity purified rabbit polyclonal antibody prepared as previously described.7 (link) Detection antibodies for the MBP1 and CLC/Gal-10 ELISAs were biotinylated using a Biotin-XX Microscale Protein Labelling Kit (B30010; Molecular Probes, Invitrogen, Eugene, Oregon, USA) according to the manufacturer's instructions. MBP1 and CLC/Gal-10 ELISA assays detected these eosinophil-derived proteins in the ranges of 11.8–750 and 0.125–16 ng/ml, respectively, with recoveries of 80%–100% and 100%–120%, signal to noise ratios >5 and coefficient of variation (CV) of 15%.
Specific parameters and detailed protocol and methods associated with the EPX-based ELISA in mice have been recently described.23 (link) EPX ELISA detected EPX in the range of 8–1024 ng/ml, with recovery of 90%, signal to noise ratios >15 and an assay CV of 13%.
20–22 For MBP1, mouse monoclonal antibodies were used for both capture and detection (mouse anti-human MBP1-clones, J14-8A2 and J13-6B6, respectively, kindly provided by Dr Hirohito Kita, Mayo Clinic, Rochester, Minnesota, USA), the detection antibody being biotinylated and detected using streptavidin-conjugated horseradish peroxidase (ExtraAvidin Peroxidase, E2886; Sigma-Aldrich, St. Louis, Missouri, USA) and FAST OPD Tablet substrate (P9187; Sigma-Aldrich). CLC/Gal-10 ELISAs used a mouse monoclonal capture antibody (Anti-human Gal-10/CLC, Cell Sciences, Canton, Massachusetts, USA), whereas the detection antibody was a CLC/Gal-10 affinity purified rabbit polyclonal antibody prepared as previously described.7 (link) Detection antibodies for the MBP1 and CLC/Gal-10 ELISAs were biotinylated using a Biotin-XX Microscale Protein Labelling Kit (B30010; Molecular Probes, Invitrogen, Eugene, Oregon, USA) according to the manufacturer's instructions. MBP1 and CLC/Gal-10 ELISA assays detected these eosinophil-derived proteins in the ranges of 11.8–750 and 0.125–16 ng/ml, respectively, with recoveries of 80%–100% and 100%–120%, signal to noise ratios >5 and coefficient of variation (CV) of 15%.
Specific parameters and detailed protocol and methods associated with the EPX-based ELISA in mice have been recently described.23 (link) EPX ELISA detected EPX in the range of 8–1024 ng/ml, with recovery of 90%, signal to noise ratios >15 and an assay CV of 13%.
